The aging of human skin is accompanied by increasing formation of wrinkles and reducing elasticity and strength. A distinction is made in the ageing process between intrinsic and extrinsic skin ageing. Intrinsic ageing includes natural changes to the skin, which are regulated by genetic makeup. The term extrinsic skin ageing stands for premature ageing processes brought about by exogenous influences such as sunlight, environmental pollutants (for example ozone, tobacco smoke, etc.), psychological stress and chronic inflammation. Ultraviolet radiation is the most important exogenous pathogen leading to premature ageing of the human skin (light ageing). In addition to natural sunlight, irradiation of the human skin with artificial UV radiation (solarium) is playing an ever greater role.
The structural changes responsible for the clinical image of aged skin take place primarily in the dermis. Elasticity and strength of the skin are determined substantially by the two main constituents of the dermal extracellular matrix, the two fibre proteins collagen and elastin. The dermis contains mainly collagen-1 (90-85%), which is formed exclusively from dermal fibroblasts, and, in significantly smaller amounts (10-15%), collagen-3. Elastin, the principal component of the elastic fibres of the skin apart from fibrillin, is contained in the dermis in a proportion of around 1-3%.
In comparison to young skin, old skin is characterised by a lowering concentration of collagen and elastin. This age-related loss of tissue is at least partially caused by an imbalance between activation and inhibition of proteolytic activity. An important role is played here by matrix metalloproteinases, a group of enzymes which are able to break down the macromolecules in the extracellular matrix (ECM) proteolytically. Thus it has been found that the content of MMPs is markedly higher in old skin than in young skin (J. H. Chung et al., J. Invest. Dermatol, 2001, 117, 1218-1224).
MMPs have a broad, often overlapping, substrate specificity, and when combined they are capable of destroying all protein components of the extracellular matrix. Some 20 MMPs have been identified to date. They are generally secreted as inactive pro-enzymes (pro-MMP). Of particular importance in the human skin are, above all, MMP-1 (collagenase-1), MMP-2 (gelatinase A), MMP-9 (gelatinase B) and MMP-3. In addition to collagen-1 and -3, MMP-1 also cleaves pro-MMP-2 and pro-MMP-9, thereby causing them to be activated. MMP-2 and MMP-9 belong to the elastin-degrading proteases (A. Thibodeau, Cosmetics & Toiletries 2000, 115 (11), 75-82).
MMPs also play a critical role in the premature skin aging caused by exogenous factors. Thus, an even higher level of MMPs was detected in light-aged skin as compared with aged skin protected from the light (J. H. Chung et al., J. Invest. Dermatol, 2001, 117, 1218-1224). The induction of matrix metalloproteinases was detected both for UVA and UVB and for infrared radiation. This induction was able to be observed both in vitro on cultivated human dermal fibroblasts and in vivo on UV-irradiated human skin. Stimulation with tobacco smoke also led in human dermal fibroblasts to an induction of the expression of MMP-1 and -3 (J. Krutmann, Hautarzt 2003, 54, 809-817).
The regulation of MMP activity can occur at three levels: at a transcription level, in the conversion of pro-MMP to the active form or by inactivation of MMPs by inhibitors.
The reproduction of the imbalance in the proteolytic activity of MMPs, in particular MMP-1, -2, -3 and -9, brought about by intrinsic and extrinsic skin ageing is thus an important objective in the development of new cosmetic active ingredients against skin ageing and wrinkles.
The use of MMP-1-inhibiting substances (retinyl palmitate, propyl gallate, precocene, 6-hydroxy-7-methoxy-2,2-dimethyl-1(2H)-benzopyran, 3,4-dihydro-6-hydroxy-7-methoxy-2,2-dimethyl-1(2H)-benzopyran) to prevent sunlight- and/or heat-induced ageing of the human skin was described in the laid-open specification WO 01/74320.
Matrix metalloproteinases are also significant in pathological changes to the periodontium. Periodontitis is an inflammation of the periodontium, in other words the tissues that surround and support the teeth. The periodontium comprises various tissues: the gum epithelium (gingiva), the connective tissue of the gingiva, the periodontal ligament (desmodontium), the cementum and the surrounding alveolar bone. The desmodontium is located between the surface of the root and the alveolar bone. It is a cell-rich connective tissue which holds the tooth in the bony tooth socket, the alveolus. 53 to 74% of the periodontal space is made up of collagen and oxytalan fibre bundles. The portion of the periodontal fibres incorporated into the cementum and the alveolar bone holds the tooth in the alveolus. The main clinical features of periodontitis include inflammation of the gums, attachment loss, formation of periodontal pockets and degradation of the alveolar bone.
The main cause of periodontitis is plaque. This consists of certain components of saliva, food residues and above all bacteria and their decomposition products. This special form of an infectious disease is caused in most cases by Porphyromonas gingivalis, Bacteroides forsythus and Actinobacillus actinomycetemcomitans. The continuous release of bacterial toxins, especially of lipopolysaccharides, presumably triggers the distribution of proinflammatory mediators, such as IL-1beta, TNF-alpha and PGE2 for example, in the patient's affected tissues. These signal substances stimulate the infiltration of immunocompetent cells into the populated tissue. The migration of neutrophilic granulocytes and macrophages then subsequently leads to inflammation of the gums (gingivitis) and to the release of proinflammatory mediators such as IL-1 and IL-6, for example. These in turn activate in the skin and mucous membranes the synthesis of matrix-degrading metalloproteinases (matrix metalloproteinases, MMPs), which destroy the extracellular matrix of the surrounding connective tissue. This allows bacteria, which initially interacted with the free gingiva, to penetrate further into the underlying connective tissue, continuing inflammation processes and the synthesis of MMPs there and finally loosening the connection between the uppermost layer of the epithelium and the root of the tooth. A gingival pocket is formed as a consequence. The reaction of the body is the inflammation of the gingiva and the periodontium with damage to the alveolar bone. In the final stage of periodontitis the affected person is at risk of a massive loss of teeth.
Studies (T. Kuboto et al., Arch. Oral. Biol. 1996, 41, 253-262; A. L. Ejeil et al., J. Periodontol. 2003, 74, 188-195) have shown that the levels of a series of matrix metalloproteinases (MMP-1, -3, -8, -9 and -13) are significantly higher in patients with inflammation of the gums than in patients with healthy gums. The level correlates with the severity of the gingivitis or periodontitis. Furthermore, collagen fibres decrease significantly as the inflammation of the gums becomes more marked. MMP-9 clearly acts as a marker here at an early stage of periodontitis (A. L. Ejeil et al., J. Periodontol. 2003, 74, 188-195).
MMPs also have an important role to play in the development of caries and non-caries-related losses of hard tooth structure, such as erosions for example. The teeth are constructed mainly from a bonelike substance called dentine. In the area of the crown which protrudes from the gum, the dentine is covered with the protective enamel. Dentine is made up of around 30% of a cell-free basic substance consisting largely of glycoproteins. Collagen fibres and inorganic components are incorporated herein.
The development of caries and erosions is accompanied by the demineralisation of the teeth. Mineral substances are critically responsible for the hardness of the tooth. The formation of acids by oral bacteria after consumption of sugary foods on the one hand, but also through frequent contact with highly acidic drinks (e.g. fruit juices) and highly acidic food (citrus and tropical fruits, pineapple, etc.) leads to demineralisation of the enamel and, if it continues, of the dentine too. The demineralised dentine is susceptible to degradation. It has been shown in vitro that the degradation of the organic matrix is necessary for the development of a cavity in the tooth. Tjäderhane et al. (J. Dent. Res. 1998, 77, 1622-1629) detected MMP-2, MMP-8 and MMP-9 in caries lesions and established that these are activated by acids.
Pashley et al. (J. Dent. Res. 2004, 83, 216-221) showed that even in the absence of bacteria, degradation of collagen fibres in the organic matrix occurs in dentine which has been partially demineralised by acid, due to collagenolytically active proteases. The degradation of the collagen fibres was prevented by the addition of chlorhexidine or protease inhibitors (MMP inhibitor: benzamidine hydrochloride, cysteine proteinase inhibitor: N-ethyl maleimide, epsilon-amino-n-hexanoic acid, serine protease inhibitor: phenylmethylsulfonyl fluoride).
Maintaining the health or slowing the degradation of the connective tissue of the periodontium and the collagen fibres of the teeth by preventing damage due to MMPs is therefore an important aim in the development of new active ingredients for the area of oral care and oral hygiene. In order to stop the processes described effectively, the damage caused by MMPs and in particular here by MMP-1 (collagenase-1), MMP-2 (gelatinase A), MMP-8 (collagenase-2) and MMP-9 (gelatinase B) must be inhibited at a very early stage.
The use of synthetic MMP inhibitors in periodontal diseases has been described in several publications (M. E. Ryan et al., Curr. Opin. Periodontal. 1996, 3, 85-96; R. Gendron et al., Clin. Diagn. Lab. Immunol. 1999, 6, 437-439).
A number of plant extracts have also already been described as inhibitors of various MMPs. For instance, numerous plant extracts, and the inhibition of various proteases, including several MMPs, that can be achieved with them, are described in laid-open specification WO 02/069992 A1, although no conclusions can be drawn as to the concentrations of extract used and the precise extraction conditions. From the results tables reproduced in the cited laid-open specification, no correlation can be identified between a specific plant family or a specific plant species and the effectiveness of a corresponding plant extract in the inhibition of metalloproteinases.
In Ann. Pharmaceutiques Françaises 1990, 48, 335-340 and in the European patent application EP 0283349 A, J. L. Lamaison describes the inhibition of elastase (porcine pancreatic elastase) and collagenase with extracts of plants selected from the rosaceae group and attributes the inhibition to the tannins they contain.
Ethanol and acetone extracts from Rubus fruticosus and other plants, which are to be used to prevent skin ageing, are known from Japanese laid-open specification JP 2003-160433. Production of the extracts is a lengthy process, however (extraction period: 1 week).
In the Commission E monograph, blackberry leaves in the form of aqueous tea infusions and mouthwashes are indicated inter alia for the area of application of mild inflammations of the mucous membranes of the mouth and pharynx (Bundesanzeiger, 1 Feb. 1990, issue number 22a, no. 01071). The effectiveness is attributed to the astringent effect of the tannins they contain. No mention is made, however, of the treatment of damage to the connective tissue of the periodontium and to the collagen fibres of the teeth caused by MMPs.